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High-Resolution Imaging of STIM/Orai Subcellular Localization Using Array Confocal Laser Scanning Microscopy

  • Andras T. Deak
  • Benjamin Gottschalk
  • Emrah Eroglu
  • Rene Rost
  • Markus Waldeck-Weiermair
  • Wolfgang F. Graier
  • Roland Malli
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1843)

Abstract

The expression of chimeras that consist of a fluorescent protein (FP) conjugated with a protein of interest provides the ability to visualize, track, and quantify the subcellular localization and dynamics of specific proteins in biological samples. Array confocal laser scanning microscopy is an eminently suitable technique for live-cell imaging of FP-tagged fusion proteins. Here, we describe real-time monitoring of the subcellular dynamics of the stromal-interacting molecule 1 (STIM1) and Orai1, the key protagonists of store-operated Ca2+ entry (SOCE) under resting conditions, and upon Ca2+ mobilization from the endoplasmic reticulum (ER).

Key words

Ca2+ signaling Cell transfection Fluorescence microscopy Fluorescent proteins Genetically encoded tools Image analysis Live-cell imaging 

Notes

Acknowledgments

We thank Sandra Blass and Anna Schreilechner for their excellent technical assistance and Dr. C.J.S. Edgell (University of North Carolina, Chapel Hill, NC, USA) for the EA.hy926 cells. This work was supported by the Austrian Science Funds (FWF) with the project number P 28529-B27 and also sponsoring the DKplus Metabolic and Cardiovascular Disease (W1226-B18) of the Medical University of Graz. A.T.D., B.G., and E.E. was/are a fellow/fellows of the Doctoral College “Metabolic and Cardiovascular Disease” at the Medical University of Graz and was/are funded by the FWF (A.T.D.), Nikon Austria (B.G.), or BioTechMed (E.E.) (both within the Nikon-Center of Excellence, Graz) within the Doctoral College “Metabolic and Cardiovascular Disease” (FWF W 1226-B18, DKplus Metabolic and Cardiovascular Disease).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Andras T. Deak
    • 1
    • 2
  • Benjamin Gottschalk
    • 1
  • Emrah Eroglu
    • 1
  • Rene Rost
    • 1
  • Markus Waldeck-Weiermair
    • 1
  • Wolfgang F. Graier
    • 1
  • Roland Malli
    • 1
  1. 1.Molecular Biology and Biochemistry, Gottfried Schatz Research CenterMedical University of GrazGrazAustria
  2. 2.Clinical Division of Nephrology, Department of Internal MedicineMedical University of GrazGrazAustria

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